|
| Status |
Public on Apr 05, 2018 |
| Title |
Dglycerol/BYGlicerol |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Damp Glycerol
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain/genotype: DampERG12 mutants
carbon source: glycerol
|
| Treatment protocol |
The cultures were centrifuged and each one was re-suspended with YPD medium pH 2.5 adjusted with sulfuric acid. After incubation for 1 h at 30°C with orbital agitation (150 rpm), the cells were recovered by centrifugation and immediately used to RNA extraction
|
| Growth protocol |
Cell from BY4741 strain and DampERG12 mutants, which express 10% of parental MVK activity, were grown in complete synthetic YNB medium containing glucose (20 g/L) in flasks at 30ºC and 150 rpm in a rotatory shaker to mid-exponential growth phase (OD600nm = 1), collected, washed in water, suspended in in the same medium containing either glucose (20 g/L) or glycerol (20 g/L) at an initial cell density of OD600 0.1 and cultivated in the same conditions until the cultures reach to OD600 0.5 (maximal OD reached by mutant cells in glycerol). Cells were recovered from 20 mL of cultures, centrifuged and stored at -80ºC until use.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA in the lysates was purified by using RNAspin Mini RNA Isolation Kit (cat. nr. 25-0500-71, GE HealthCare, USA) following manufacturer´s instructions and suspended in 25 μl nuclease-free water.
|
| Label |
Cy5
|
| Label protocol |
Synthesis of cDNA and labelling was performed by Two-Color Low Input Quick Amp Labeling Kit (cat. nr. 5190-2306, Agilent, USA), with Two-Color RNA spike-in kit for internal control (cat. nr. 5188-5279, Agilent, USA). For yeast samples, 100 ng of purified RNA was used for labelling.
|
| |
|
| Channel 2 |
| Source name |
Parental glycerol
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain/genotype: BY4741 strain
carbon source: glycerol
|
| Treatment protocol |
The cultures were centrifuged and each one was re-suspended with YPD medium pH 2.5 adjusted with sulfuric acid. After incubation for 1 h at 30°C with orbital agitation (150 rpm), the cells were recovered by centrifugation and immediately used to RNA extraction
|
| Growth protocol |
Cell from BY4741 strain and DampERG12 mutants, which express 10% of parental MVK activity, were grown in complete synthetic YNB medium containing glucose (20 g/L) in flasks at 30ºC and 150 rpm in a rotatory shaker to mid-exponential growth phase (OD600nm = 1), collected, washed in water, suspended in in the same medium containing either glucose (20 g/L) or glycerol (20 g/L) at an initial cell density of OD600 0.1 and cultivated in the same conditions until the cultures reach to OD600 0.5 (maximal OD reached by mutant cells in glycerol). Cells were recovered from 20 mL of cultures, centrifuged and stored at -80ºC until use.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA in the lysates was purified by using RNAspin Mini RNA Isolation Kit (cat. nr. 25-0500-71, GE HealthCare, USA) following manufacturer´s instructions and suspended in 25 μl nuclease-free water.
|
| Label |
Cy3
|
| Label protocol |
Synthesis of cDNA and labelling was performed by Two-Color Low Input Quick Amp Labeling Kit (cat. nr. 5190-2306, Agilent, USA), with Two-Color RNA spike-in kit for internal control (cat. nr. 5188-5279, Agilent, USA). For yeast samples, 100 ng of purified RNA was used for labelling.
|
| |
|
| |
| Hybridization protocol |
Two arrays, corresponding to biological duplicate, were used for each sample combination: parental glycerol (Cy5, Target ) x parental glucose (Cy3, reference); Damp glycerol (Cy5, Target) x Damp glucose (Cy3 reference); Damp glucose (Cy5, Target) x parental glucose (Cy3, reference); Damp glycerol (Cy5, Target) x parental glycerol (Cy3, reference). Target and reference cRNA samples were mixed and the hybridizations were performed on yeast gene expression 8x15K spots slides (cat. nr. G4813A-016322, Agilent, USA) at 65 ºC for 17 hours at 10 r.pm. in the Microarray Hybridization Oven (cat. nr. G2545A, Agilent, USA).
|
| Scan protocol |
The slides were placed into the High-Resolution Microarray Scanner (cat. nr. G2505-60502, Agilent, USA). The fluorescence data were extracted (.txt files) for each spot by using Scan Control 8.5.1 software (Agilent, USA)
|
| Data processing |
Data were then analyzed using the R statistical language with the specific LIMMA and MArray packages. Background correction was done with normexp method. Robust spline and quantile methods were used for normalization within and between arrays, respectively. A list of differentially expressed genes was generated by applying adjusted p-values for multiple tests. Groups of genes with statistical significance (Adj.p<0.05) were utilized
|
| |
|
| Submission date |
Apr 25, 2017 |
| Last update date |
Apr 05, 2018 |
| Contact name |
Marcos Antonio Morais |
| E-mail(s) |
marcos.morais@pesquisador.cnpq.br
|
| Organization name |
Federal University of Pernambuco
|
| Department |
Department of Genetics
|
| Lab |
Interdepartmental Research Group in Metabolic Engineering
|
| Street address |
Av. Moraes Rego, 1235
|
| City |
Recife |
| State/province |
Pernambuco |
| ZIP/Postal code |
50670-901 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL16244 |
| Series (1) |
| GSE98163 |
Influence of deficiency in mevalonate kinase (MVK) on wide gene expression in Saccharomyces cerevisiae |
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